The 35 year quest for a vaccine that confers protective efficacy against congenital infection with human cytomegalovirus (HCMV) remains unmet. Complexities in HCMV natural history, incompletely defined correlates of immune protection and financial and logistical factors in designing sufficiently powered clinical trials all contribute to the absence of a licensed HCMV vaccine(s). There is now increased recognition from studies in both humans and rhesus macaques (RM) that (1) the mechanism of HCMV and rhesus CMV (RhCMV) entry into cells, such as endothelial and epithelial cells, is distinct from the mechanism of fibroblast entry, and (2) seroimmune individuals develop neutralizing antibodies against the viral proteins mediating epithelial/endothelial cell tropism. Studies with HCMV show that the UL128, 131A, 130 proteins together form a complex with gH and gL that is essential for epithelial/endothelial cell tropism via an endocytic pathway, and the absence of any one of these proteins greatly restricts HCMV infection to gB-mediated entry into cells, such as fibroblasts. The preponderance of evidence from natural history and tissue culture studies evidence indicates that vaccine strategies will need to block both the endocytic (UL128 complex) and fibroblast (gB) entry pathways to establish a sufficient level of protective immunity to reduce or prevent congenital infection. The data with HCMV and our progress in both expressing a secreted form of RhCMV UL128 from rMVA as a single subunit and characterizing the role of the UL128 complex in RhCMV natural history lead to the hypothesis that vaccine strategies targeting the UL128 complex will show enhanced restriction of virus shedding in bodily fluids. Based on this hypothesis, vaccination of RhCMV-naove RM with the UL128 complex will have a significantly reduced frequency and titer of systemic infectious virus, resulting in reduced levels of shed virus. This hypothesis will be rigorously tested in our primate model of HCMV persistence and pathogenesis through the following Aims. (1) Construction and characterization of plasmid expression and MVA vectors for the UL128 complex. (2) Optimization of vaccination in macaques with expression vectors constructed in Aim 1, and characterization of peripheral and mucosal antibody responses to the UL128 complex in RhCMV-infected macaques. (3) Immunization of RhCMV-negative RM by the optimized prime/boost regimen from Aim 2 followed by either a subcutaneous or mucosal route of RhCMV challenge capable of both fibroblast and endocytic entry. PUBLIC HEALTH RELEVANCE: This proposal will show in a primate RhCMV model that faithfully recapitulates human infection and expresses the monkey equivalent of the HCMV UL128 complex that a protective vaccine augmented with RhCMV UL128 complex antigens will significantly limit the capacity of challenge virus to disseminate. These studies should lead to an optimal prophylactic HCMV clinical strategy that can lead to clinically relevant reductions in the potential for both vertical and horizontal transmission of HCMV.